The state of the art provides methods for producing antibodies in vitro (eg, using phage, ribosome or yeast display) or in vivo (eg, using non-human vertebrates (eg, mice and rats) and cells comprising transgenic immunoglobulin loci). Such in vivo systems (eg, Xenomouse™) have used completely human transgenic heavy chain loci which comprise human variable regions (human VH, D and JH gene segments) upstream of human constant regions (eg, human mu upstream of human gamma constant gene segments). Subsequently, it has been discovered that the use of totally human transgenic loci in such in vivo systems is detrimental and B-cell development is hampered, leading to relatively small B-cell compartments and restricted utility for generating antibodies. Later-generation transgenic animals (eg, the Velocimouse™) have been created which have chimaeric heavy chain loci in which a human variable region is upstream of endogenous (eg, mouse or rat) constant regions (ie, mouse mu constant region upstream of gamma constant region, in germline configuration). This enables the harnessing of endogenous control mechanisms for B-cell and antibody development, and as such the extent of problems of totally human transgenic loci are not seen. Methods of constructing transgenic vertebrates and use of these to generate antibodies and nucleic acids thereof following antigen immunisation are known in the art, eg, see U.S. Pat. No. 7,501,552 (Medarex); U.S. Pat. Nos. 5,939,598 & 6,130,364 (Abgenix); WO02066630, WO2011163311 & WO2011163314 (Regeneron); WO2011004192 & WO2011158009 (Kymab Limited); WO2009076464, WO2009143472, EP1414858, WO2009013620A2, WO2010070263A1 & WO2010109165A2 (Harbour Antibodies); EP1399559 (Crescendo Biologics) and WO2010039900 (Ablexis), the disclosures of which are explicitly incorporated herein including, but not limited to, for the purpose of providing the skilled person with guidance of how to make non-human animals bearing transgenic immunoglobulin loci and to inactivate endogenous loci expression.
The art has recognised the desirability of producing artificial combinations of epitope or antigen binding sites and specificities. So, the art has proposed multivalent antigen-binding antibodies and polypeptides in which a plurality of binding sites are synthetically combined to provide for binding to multiple epitopes on the same or different targets. When the targets are different, multispecific constructs are possible that can target and bind to a plurality of different antigens providing the epitopes (eg, to neutralise the antigens). One technique involves the in vitro combination (fusion) of different hybridomas to produce quadromas in which pairings of heavy and light chains from different antibodies are obtained (Milstein & Cuello, Nature, 1983, 305(5934): pp 537-40). This technique disadvantageously leads to mis-match pairings and multiple different combinations of antibody chains, so that the yield of the desired combination of specificities is relatively low. Subsequent techniques have used in vitro antibody engineering in order to synthetically combine epitope binding moieties (eg, antibody varirable domains, dAbs or scFvs) to produce multivalent, multispecific constructs. The Epitope binding moieties are individually selected (usually on the basis of epitope binding affinity) in in vitro techniques such as phage display, ribosome display and yeast display. Once desirable moieties have been identified, these are then combined in vitro by engineering and the performance and characteristics of the resultant multivalent constructs are assessed. While providing for multi-valency and multi-specificity, these techniques are hampered, however, because such engineering in vitro can downgrade the desirable characteristics of the resultant antibody. For example, antigen-binding affinity, antigen specificity, expressability (eg, in cell lines such as CHO or HEK293 cells), half-life and/or biophysical characteristics (eg, melting temperature, solution state, resistance to aggregation etc) can be downgraded, thereby hampering development of the antibody as drugs for human therapeutic or prophylactic use. It would be desirable to have means for producing, maturing and selecting multivalent and multispecific antibodies that addresses these shortcomings in the art. Multivalent and multispecifc formats are disclosed, for example, in Trends Biotechnol. 2010 July; 28(7):355-62. Epub 2010 May 4; “Multivalent antibodies: when design surpasses evolution”; Cuesta A M et al.
Reference is made to the patent applications cited above from Harbour Antibodies and Crescendo Biologics; these relate to the production of heavy chain-only antibodies (H2 antibodies) which lack CH1 and are devoid of light chains. In Proc Natl Acad Sci USA. 2006 Oct. 10; 103(41):15130-5. Epub 2006 Oct. 2; “Generation of heavy-chain-only antibodies in mice”; Janssens et al transgenic mice (MGΔ mice) are disclosed which comprise an antibody heavy chain locus comprising llama VHH exons and a Cμ region upstream of Cγ2 and Cγ3 regions in which CH1 has been deleted. The mice have a μMT background and express light chains. The heavy chain MGΔ loci are introduced by injection of DNA into fertilized mouse eggs follow by random integration of one to five copies of the locus into the mouse genomes. The MGΔ loci did not rescue B-cell development. MGΔ mice contained very few B-cells expressing cell-surface chimaeric Ig; 30% of the bone marrow B220+ cells expressed intracellular IgM, but no IgG. Thus, there is a need for improved transgenic heavy chain loci as well as vertebrates and cells comprising these for the generation of H2 antibodies (eg, class-switched H2 antibodies such as gamma-type H2 antibodies).